PROJECT SUMMARY Despite limited knowledge about their toxicity and health effects, the use of electronic cigarettes (e-cigs) has increased among former smokers and young adults who have never smoked. Increasing evidence shows that e-cig aerosol contains relatively high levels of toxic metals, including lead and nickel, metals with strong evidence for a role in cardiovascular (CV) disease. The objective of this proposal is to evaluate (1) the association of e-cig use with metal exposure, (2) the association of e-cig use with coronary artery calcification and other relevant pathways (endothelial cell health, blood pressure and inflammation), and (3) the potential role of e-cig metals to explain, at least in part, the CV effects of e-cigs. In preliminary studies, we found marked increases in metal levels in the generated aerosol compared to e-liquid from the dispenser from devices of daily e-cig users, demonstrating that the heating coil is a source of metal exposure. Metals in e-cig aerosols, moreover, were positively associated with metal biomarker levels. We will conduct a longitudinal study of men and women 18 to 50 years of age of diverse race/ethnic backgrounds from New York City (130 never tobacco smokers or e-cig users (controls), 130 e-cig users who are never tobacco smokers, 130 e-cig users who are former smokers, and 130 e-cig users who are current smokers and evaluate each participant 3 times over a 1.5-year period (month 1, 9, and 18). For exposure assessment, we will collect data on e-cig use and use patterns from a questionnaire and 4-weekly mobile assessments (month 3, 6, 12, 15), an aerosol sample from the participants' e-cig device and urine and blood samples to measure metals. For outcome assessment, we will measure coronary artery calcification (CAC) using cardiac CT-scan at baseline estimating the spatially weighted calcium score (SWCS). The SWCS provides a valid, continuous measure of CAC suitable for quantifying the extent of atherosclerosis without a threshold. At each visit will also measure endothelial cell health through endothelium-dependent arterial vasodilation using EndoPAT2000 device and endothelial cells- derived microparticles (EMPs) (a marker of endothelial cell injury), blood pressure, and biomarkers of inflammation (C-reactive protein and interleukin-6). We will estimate differences in metal biomarkers and in CV risk comparing e-cig users to non-users and by e-cigarette use patterns (e.g., type of device, number of puffs, e-liquid consumed per week, type of coil). In a formal mediation analysis, we will assess the association of e- cig use with the CV measures accounting for the mediating role of metals. The proposed longitudinal study utilizes interdisciplinary expertise in the characterization of CV disease, assessment of metals, the recruitment and examination of e-cig users, and the general field of tobacco regulatory science, to fill a critical gap in the knowledge about the possible long-term effects of e-cig use and to help inform e-cig regulatory action.